Murine polyomavirus provides a valuable model for neoplastic transformation. The virus causes a broad spectrum of tumors in animals. This means that leads developed in the laboratory can be readily tested in the host. Studies on polyoma have repeatedly provided insight into basic mechanisms of cellular growth regulation. Tyrosine kinase signaling and phosphatidylinositol 3-kinase (PI3K) signaling are two fundamental mechanisms uncovered from studies on polyoma. Middle T antigen (MT) is the major transforming protein of polyomavirus. It is always necessary, and often sufficient, for cellular transformation by polyoma. This subproject will extend our knowledge of MT and its signaling based on leads developed in the previous project period. There are 4 specific aims. 1) We have shown that MT activates the small GTPase Ral. We will examine how MT activates Ral. We will test the role of Ral in MT transformation. Since Ral functions in trafficking processes including secretion, we will test MT function there. 2) MT induces osteopontin, a secreted protein thought to be linked to transformation and metastasis. We will determine how MT regulates osteopontin. We will determine the role of osteopontin in MT transformation by examining tumor profiles. Using breast cancer and hemangioma models, we will test the role of osteopontin in metastasis and in cell recruitment. We will initiate structural analysis of MT using NMR to determine the structure of the portion of MT that interacts with downstream targets such as Shc and PI3K. We will then map the interaction surface between MT and p85 of PI3K or Shc. Finally, there is strong genetic evidence suggesting that there are undiscovered MT binding proteins. We will use tandem affinity purification and mass spectrometry to identify them. We will determine the MT sequences required for their interaction. We will test their function in MT assays for transformation.